Initiation of human DNA replication entails a highly regulated assembly of polymerase complexes along with chaperone proteins and other enzymes. The polymerase alpha with its associated primase activity is the only polymerase involved in this initiation process. Our research is concentrated on understanding the regulation and mechanism of the human DNA polymerase alpha - primase complex. This enzyme complex is composed of four proteins, a 180 kDa phospho-glyco protein containing the DNA polymerase activity, a 70 kDa phosphoprotein with no known function, and two smaller subunits of 49 and 58 kDa containing the primase activity. Current research is focused on the enzymatic mechanism and regulation of the two primase subunits. In the mouse p49 primase subunit fourteen site specific alanine mutations were produced to locate the active site and to address the roles of highly conserved charged residues within the enzyme. These mutant enzymes have been purified and extensively characterized by steady state kinetics and biophysical assays. The analysis of these mutants identified three aspartic acid residues which bind metals essential for catalytic activity. In addition, a pair of arginine residues was identified which are important in the processivity of the enzyme and in binding the incoming ribonucleotides. In order to understand the human DNA replication machinery the cDNA's for these two human DNA primase subunits were isolated, sequenced and overexpressed in E. coli. Specific assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the presence of the larger subunit. A two plasmid system was developed for the co-expression of both subunits in E. coli. This system was exploited to express and study deletion constructs of the larger, Hp58 subunit to investigate its role in primer formation. Analysis of the complexes formed between the deleted Hp58 constructs and the native Hp49 subunit revealed that protein-protein contacts between the two subunits are made over several domains of the Hp58 subunit. Of four primase complexes containing different Hp58 deletions only one complex was able to support initiation as measured by the formation of dinucleotides. All complexes supported the extension of oligoA primed polydT suggesting that the intrinsic RNA polymerase activity residing in the smaller subunit was not affected. These results implicate several regions of the Hp58 subunit in contact with the Hp49 subunit in the initiation of primers during DNA replication.